Team:UC Davis/Protocols
From 2011.igem.org
(Difference between revisions)
Line 229: | Line 229: | ||
</div> | </div> | ||
</div> | </div> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
</html> | </html> | ||
Revision as of 20:36, 27 September 2011
Start a Family
Got a favorite BioBrick? Check our our process for expanding basic parts into part families.Criteria
View our judging criteria for iGEM 2011 here.
Protocols
Here, you will find protocols for the procedures we use in our lab. These have been passed on by senior members of the lab in which we work, taken from OpenWetWare.org, or adapted from reference papers by members of our team.
You may select any of these protocols to navigate directly to it on this page:
Restriction Enzyme Double Digest
Gel Extraction and Purification
Transformations
Liquid Cultures
PCR
Minipreps
Ligation
Error-Prone PCR
Nanodrop
Glycerol Stocks
PCR Purification
You may select any of these protocols to navigate directly to it on this page:
Restriction Enzyme Double Digest
Gel Extraction and Purification
Transformations
Liquid Cultures
PCR
Minipreps
Ligation
Error-Prone PCR
Nanodrop
Glycerol Stocks
PCR Purification
Restriction Enzyme Double Digest
Materials:
- 22 uL dH2O
- 1 uL BSA
- 5 uL Buffer
- 20 uL Template
- 1 uL Enzyme 1
- 1 uL Enzyme 2
Buffer Compatibility Chart:
Number indicates percent activity of enzyme in given solution.
1 | 2 | 3 | 4 | |
---|---|---|---|---|
EcoRI | 100 | 100 | 100 | 100 |
SpeI | 75 | 100 | 25 | 100 |
PstI | 75 | 75 | 100 | 50 |
NheI | 100 | 100 | 10 | 100 |
XbaI | 0 | 100 | 75 | 100 |
Procedure
- Mix reactants thoroughly.
- Place at 37 C for 3 hours.
- Increase to 80 C for 20 minutes to kill enzymes (some enzymes need only a 65 C heatkill, check enzyme).
- Run on a gel and extract product.
Gel Extraction and Purification
Materials
- GeneJET Gel Extraction Kit
- Binding Buffer (1 uL for every mg of agarose gel)
- 700 uL of Wash Buffer
- 50 uL of Elution Buffer
Procedure
- Add the binding buffer to the gel slice in a microcentrifuge tube.
- Incubate the gel mixture at 50-60 °C for 10 minutes (until melted).
- Transfer the solution to a GeneJET purification column.
- Centrifuge for 30-60 seconds at 12000 x g and discard the flow through.
- Add Wash Buffer and centrifuge for 1 minute.
- Discard flow through, then centrifuge empty column for 1 minute.
- Transfer the column into a fresh 1.5 ml microfuge tube.
- Add Elution Buffer.
- Centrifuge for 1 minute and collect the flow-through.
Transformations
Materials
- Competent cells
- DNA template
- 800 uL of LB
- LB+antibiotic plates
Procedure
- Thaw competent cells on ice.
- Transfer 50 uL of competent cells to chilled falcon tubes.
- Add 1 uL of template to cells (2.5 uL if dilute).
- Incubate on ice for 30 minutes.
- Heat schock in 42 °C water bath for 90 seconds.
- Immediately place back onto ice for 2 minutes.
- Add 800 uL of LB to each tube.
- Incubate at 37 °C for 1 hour.
- Place 200 uL of the transformed cells on plates containing LB and the appropriate antibiotic.
- Incubate overnight at 37 °C.
Preparing Liquid Cultures
Materials
- LB
- Plated colonies of cells
- Antibiotic stock
Procedure
- Working near a flame or in a laminar flow hood, add 5 mL of LB to each falcon tube.
- Add the appropriate amount of antibiotic.
- For example, 5 uL of 35 mg/mL chloramphenicol stock or 10 uL of 100 mg/mL carbenicillin stock.
- With a tip, scoop a colony from your agar plate and place it in the falcon tube.
- Incubate overnight at 37 °C.
PCR
Mix
- 10ul Q solution
- 5ul 10x buffer
- 1.25ul DNTPs
- 1ul Forward primer
- 1ul Reverse primer
- 1ul Template
- .3ul Taq
- .15ul PFU
- 30ul dH2O
Minipreps
Materials
- Liquid culture
- Miniprep kit (QIAprep Spin Miniprep Kit)
Procedure
- Centrifuge liquid culture of cells.
- Discard the supernatant.
- Resuspendd the pelleted cells in 250 uL of Buffer P1 and transfer to a microcentrifuge tube.
- Add 250 uL of Buffer P2.
- Invert 4-6 times until the solution become clear.
- Add 350 uL Buffer N3.
- Invert 4-6 times.
- Centrifuge for 10 minutes at 17900xg.
- Apply the supernatant to a spin column.
- Centrifuge for 1 minute and discard the flow-through.
- Wash the spin column with 0.5 ml Buffer PB.
- Centrifuge for 1 minute and discard the flow through.
- Wash the spin column with 0.75 ml Buffer PE.
- Centrifuge for 1 minute and discard the flow through.
- Centrifuge an additional 1 minute to remove residual wash buffer.
- In a clean 1.5 ml microcentrifuge tube, elute DNA with 50 ul Buffer EB.
- Centrifuge for 1 minute and collect the flow-through.
Ligation
Materials
- Digested vector
- Digested insert
- Water
- T4 DNA ligase.
- T4 DNA ligase buffer.
Procedure
- Mix these materials in the amounts determined by the reaction volume calculator.
media:UC_Davis_Reaction_Volume_Calculator.xls
Error-Prone PCR
Mix
- 5ul 10x buffer
- 2.5ul Unbalanced DNTPs
- 1ul Forward primer
- 1ul Reverse primer
- 10uL 25 mM MgCl2
- 7.5uL 1 mM MnCl2
- 0.1ul Template
- .5ul Taq
- 22.4ul dH2O
Unbalanced DNTP Mix
- 20uL dATP
- 20uL dGTP
- 100uL dCTP
- 100uL dTTP
- 260uL H2O
(Store at -20 C)